Method of producing natural killer cells and composition for treating cancer

ABSTRACT

A method for producing natural killer cells is disclosed. The method comprises isolating peripheral blood mononuclear cells (PBMCs) from a blood sample; isolating at least one of CD56+ cells and/or CD3−/CD56+ cells from the PBMCs; and co-culturing the at least one of CD56+ cells and/or CD3−/CD56+ cells with a combination of feeder cells in the presence of a cytokine. A composition for treating cancer is also disclosed. The composition comprises the CD56+ natural killer cells produced by the disclosed method and a cytokine.

INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

This application claims the benefit of Korean Patent Application No.KR-10-2018-0012938, filed Feb. 1, 2018, Korean Patent Application No.KR-10-2018-0012942, filed Feb. 1, 2018, Korean Patent Application No.KR-10-2019-0001981, filed Jan. 7, 2019, and Korean Patent ApplicationNo. KR-10-2019-0001983, filed Jan. 7, 2019, the disclosures of which arehereby incorporated by reference in their entireties.

BACKGROUND Field

The present disclosure relates to a manufacturing method for high-puritynatural killer cells.

Description of the Related Art

The human body is protected from pathogens by an immune response,coordinated by the immune system, which is composed of manyimmune-related cells, chemical mediators, such as cytokines, and thelike. Leukocytes, especially lymphocytes, play an important role in suchan immune system. Lymphocytes are involved in both innate and acquiredimmunity.

Natural killer cells (NK cells) are one type of innate immune cells,which are known to non-specifically kill cancer, recognize and killviruses, bacteria, and the like, and kill pathogens with enzymes such asperforin and granzyme or by Fas-FasL interaction. In the case of cancerpatients, it has been reported that a decrease in cancer cellcytotoxicity of these NK cells is associated with the onset of varioustypes of cancer, such as lung cancer (Carrega P, et al., Cancer, 2008:112: 863-875), liver cancer (Jinushi M, et al., J Hepatol., 2005: 43;1013-1020), breast cancer (Bauernhofer T, et al., Eur J Immunol., 2003:33: 119-124), uterine cancer (Mocchegiani E., et al., Br j Cancer.,1999: 79: 244-250), blood cancer (Tajima F., et al, Lekemia 1996: 10:478-482), and the like. Accordingly, for cancer therapy, it is desirableto increase the cancer cell cytotoxicity of the NK cells.

In order to obtain the therapeutic effect of NK-mediated killing of thecancer cells, a large amount of NK cells having high purity is required,but it is not easy to obtain a large amount of blood from the cancerpatient, and of the proportion of NK cells in the blood is small, onlyabout 5 to 20%. Thus, it has been difficult for using the NK cells as animmunotherapeutic agent.

As a result, it is desirable to effectively expand and proliferate onlythe NK cells, but in a conventional method of proliferating NK cells,various expensive cytokines need to be used at a high concentration,thus the corresponding therapy is only available to some financiallystable patients. Further, according to conventional methods ofproliferating NK cells, other types (e.g., T cells, B cells, etc.) ofimmune cells may be present together with the NK cells, and allogenicadministration of the NK cells containing T cells may cause a graftversus host disease (GVHD) and allogenic administration of the NK cellscontaining B cells to blood-type incompatible subjects may cause apassenger B-lymphocyte syndrome, and thus, the anti-cancer effect is notmaximized.

Further, in addition to expanding and proliferating NK cells, it isdesirable to highly maintain the functions of NK cells until theexpanded and proliferated NK cells are actually used. As a result, thedevelopment of a composition capable of promoting the proliferation ofthe NK cells, increasing production of cytokines such as TNF-, INF- andGM-CSF derived from the NK cells, and increasing cancer cellcytotoxicity of the NK cells is sought.

SUMMARY

This application is related to methods of producing high-purity naturalkiller cells, and a cell therapeutic composition for treating cancercomprising high-purity natural killer cells and cytokines. Any features,structures, or steps disclosed herein can be replaced with or combinedwith any other features, structures, or steps disclosed herein, oromitted. Further, for purposes of summarizing the disclosure, certainaspects, advantages, and features of the inventions have been describedherein. It is to be understood that not necessarily any or all suchadvantages are achieved in accordance with any particular embodiment ofthe inventions disclosed herein. No individual aspects of thisdisclosure are essential or indispensable.

In an embodiment, a method of producing natural killer cells isdisclosed. The method includes: isolating peripheral blood mononuclearcells (PBMCs) from a blood sample; isolating at least one of CD56+ cellsand/or CD3−/CD56+ cells from the PBMCs; and co-culturing the at leastone of CD56+ cells and/or CD3−/CD56+ cells with a combination of feedercells in the presence of a cytokine.

In certain embodiments, isolating at least one of CD56+ cells and/orCD3-/CD56+ cells from the PBMCs is conducted by using at least one ofCD56 microbeads and CD3 microbeads. In certain embodiments, the cytokineis selected from a group consisting of IL-2, IL-21, IL-15, Flt3-L, SCF,IL-7, IL-18, IL-4, type I interferons, GM-CSF, IGF 1, and combinationsthereof. In certain embodiments, the cytokine may be added at aconcentration of 50-1000 IU/mL.

In certain embodiments, the combination of feeder cells includesirradiated Jurkat cells and irradiated Epstein-Barr virus transformedlymphocyte continuous line (EBV-LCL) cells. In a variation, the ratio ofthe irradiated Jurkat cells and the irradiated EBV-LCL cells may beabout 1:0.1-5. Each of the irradiated Jurkat cells and the irradiatedEBV-LCL cells may be obtained by irradiation of 50-500 Gy.

In certain embodiments, the co-culturing may include co-culturing for1-50 days.

In certain embodiments, the method may further include co-culturing theat least one of CD56+ cells and/or CD3−/CD56+ cells with a combinationof feeder cells, in the presence of a first cytokine for a first period;and subsequently co-culturing the at least one of CD56+ cells and/orCD3−/CD56+ cells with the combination of feeder cells, in the presenceof a second cytokine for a second period. In a variation, the secondcytokine may be added once or more during Day 0-6 of the second period.The second cytokine may be added once or more during the first six daysof every fourteen-day cycle during the second period. The first cytokinemay be IL-2. The second cytokine may be IL-21. The second cytokine maybe added at a concentration of 10-100 ng/mL.

In certain embodiments, the at least one of CD56+ cells and/orCD3−/CD56+ cells and the combination of feeder cells is co-cultured witha ratio of about 1:1-100 of CD56+ cells and/or CD3−/CD56+ cells tofeeder cells.

In certain embodiments, a composition made by the method is disclosed.

In an embodiment, a composition for treating cancer in a patient in needthereof is disclosed. The composition includes: an effective amount ofCD56+ natural killer cells derived from peripheral blood, wherein theeffective amount is in a range of about 1×10⁶ to 5×10⁸ cells per kg ofthe patient's body weight, and wherein the CD56+ natural killer cellsare at least about 90% pure; IL-2 having a concentration of 50-50,000IU/mL; and a pharmaceutically acceptable carrier.

In certain embodiments, the cytokine may be selected from a groupconsisting of IL-2, IL-21, IL-15, Flt3-L, SCF, IL-7, IL-18, IL-4, type Iinterferons, GM-CSF, IGF 1, and combinations thereof. In a variation,the cytokine may be IL-2. The cytokine may have a concentration of50-50,000 IU/mL.

In certain embodiments, the cancer is selected from a group consistingof: blood cancer, stomach cancer, pancreatic cancer, cholangiocarcinoma,colon cancer, breast cancer, liver cancer, ovarian cancer, lung cancer,kidney cancer, prostate cancer and neuroblastoma.

In certain embodiments, the composition includes less than about 1% Tcells.

BRIEF DESCRIPTION OF THE DRAWINGS

Various embodiments are depicted in the accompanying drawings forillustrative purposes, and should in no way be interpreted as limitingthe scope of the embodiments. Furthermore, various features of differentdisclosed embodiments can be combined to form additional embodiments,which are part of this disclosure.

FIG. 1A illustrates a graph showing cell growth rates of NK cellsproduced from PBMCs, CD56+ cells, and CD3−/CD56+ cells.

FIG. 1B illustrates graphs showing cell growth rates of NK cellsproduced from PBMCs and CD56+ cells with or without treating with IL-21.

FIG. 2 illustrates a graph showing the purity of CD3−/CD56+NK cellsproduced from PBMCs or CD56+ cells with or without treating with IL-21.

FIG. 3 illustrates a plate design for analyzing anticancer activity ofNK cells.

FIG. 4A illustrates graphs showing the short-term cytotoxicity of NKcells produced from PBMCs and CD56+ cells for various effectorcell:target cell (E:T) ratios.

FIG. 4B illustrates graphs showing the long-term cytotoxicity of NKcells produced from PBMCs and CD56+ cells against AGS, A549, andMDA-MB-231 cells.

FIGS. 5A-5B illustrate graphs showing cell growth rates of NK cellsproduced by treating with IL-21 during various periods.

FIG. 6 illustrates graphs showing cell growth rates of NK cells producedby treating with IL-21 with various concentrations.

FIG. 7A illustrates a graph showing the short-term cytotoxicity of NKcells produced by treating with 11-21 during various periods for variousE:T ratios.

FIGS. 7B-7C illustrate graphs showing the long-term cytotoxicity of NKcells produced by treating with IL-21 during various periods againstAGS, A549, and MDA-MB-231 cells.

FIG. 8A illustrates graphs showing the short-term cytotoxicity of NKcells produced by treating with IL-21 with various concentrations forvarious E:T ratios.

FIG. 8B illustrates graphs showing the long-term cytotoxicity of NKcells produced by treating with IL-21 with various concentrationsagainst AGS, A549, and MDA-MB-231 cells.

FIG. 9 illustrates graphs showing cell growth rates of NK cells withfeeder cell stimulations.

FIG. 10A illustrates graphs showing cell growth rates of NK cellsproduced from PBMCs of a cancer patient with or without treatment ofL-21.

FIG. 10B illustrates a graph showing purity of CD3−/CD56+NK cellsproduced from PBMCs of a cancer patient with or without treatment ofIL-21.

FIG. 10C illustrates a graph showing the short-term cytotoxicity of NKcells produced from PBMCs with or without treatment of IL-21 againstK562 cells.

FIG. 10D illustrates graphs showing the long-term cytotoxicity of NKcells produced from PBMCs with or without treatment of IL-21 againstAGS, A549, and MDA-MB-231 cells.

FIG. 11 illustrates graphs showing survival rate of NK cells treatedwith or without IL-2.

FIG. 12 illustrates graphs showing the cytotoxicity of NK cells treatedwith or without IL-2 against various cancer cells at various E:T ratios.

FIG. 13 illustrates photographs of remaining NIH:OVCAR-3 cells treatedwith NK cells treated with or without IL-2.

FIG. 14 illustrates photographs of remaining AGS cells treated with NKcells treated with or without IL-2.

DETAILED DESCRIPTION

A method for producing high-purity NK cells without using expensivecytokines has been developed by the inventors. The inventors found that,after CD56+ cells are isolated from peripheral blood mononuclear cells,when the CD56+ cells isolated from peripheral blood mononuclear cellsare co-cultured with feeder cells in the presence of cytokines,high-purity CD56+NK cells could be produced. Also, the present inventorshave developed a cell therapeutic composition for treating cancercomprising NK cells which are effectively usable for allogenic therapy.As a result, the inventors found that when a specific cytokine was addedto CD56+NK cells isolated from peripheral blood mononuclear cells, highsurvival rate and high anti-cancer activity were exhibited. Therefore,the inventors sought to develop a method for expanding NK cells and toprovide a cell therapeutic composition for the treatment of cancercomprising expanded peripheral blood-derived CD56+NK cells together withcytokines.

According to some embodiments, a method for producing high-purity NKcells may include: isolating peripheral blood mononuclear cells (PBMCs)from a blood sample (“First Isolation Step”); isolating cells selectedfrom a group consisting of CD56+ cells and CD3−/CD56+ cells from theperipheral blood mononuclear cells (“Second Isolation Step”); andco-culturing the cells selected from a group consisting of CD56+ cellsand CD3−/CD56+ cells together with feeder cells in the presence ofcytokine (“Culturing Step”). Each step is described in greater detailherein. The CD3−/CD56+ cells produced according to the disclosed methodmay exhibit not only higher purity and higher anti-cancer activity, butalso other distinguished characteristics, such as having differentsurface markers or activated receptors, for example, one or more fromCD16, CD25, CD27, CD28, CD69, CD94/NKG2C, CD94/NKG2E, CD266, CD244,NKG2D, KIR2S, KIR3S, Ly94D, NCRs, IFN-α, IFN-b, CXCR3, CXCR4, CX3CR1,CD62L and CD57, as compared with NK cells produced from peripheral bloodmononuclear cells without isolating CD56+ cells.

First Isolation Step

In the present specification, the “blood sample” may be, but not limitedto, whole blood of the peripheral blood or leukocytes isolated from theperipheral blood using leukapheresis. Further, the peripheral blood maybe obtained from a normal person, a patient having a risk of cancer, ora cancer patient, but the source of the peripheral blood is not limitedthereto.

In the present specification, the term “leukapheresis” may refer to amethod of selectively removing (isolating) leukocytes from the collectedblood and then giving the blood to a patient again, and in someembodiments, the leukocytes isolated by the method may be used withoutadditional methods such as a Ficoll-Hypaque density gradient method.

In the present specification, the term “peripheral blood mononuclearcell” may be used interchangeably with “PBMC”, “mononuclear cell” or“monocyte”, and may refer to a mononuclear cell isolated from theperipheral blood which is generally used for anti-cancer immunotherapy.The peripheral blood mononuclear cells may be obtained from thecollected human blood using known methods such as a Ficoll-Hypaquedensity gradient method.

In some embodiments, the peripheral blood mononuclear cells may beautologous, but allogenic peripheral blood mononuclear cells may also beused for producing high-purity NK cells for anti-cancer immunotherapyaccording to methods described herein. Further, in some embodiments, theperipheral blood mononuclear cells may be obtained from a normal person,but the peripheral blood mononuclear cells may be also obtained from apatient having a risk of cancer and/or a cancer patient.

In the present specification, the term “CD56+ cells” may be usedinterchangeably with “CD56+NK cells”, or “CD56+ natural killer cells”,and the term “CD3−/CD56+ cells” may be used interchangeably with“CD3−/CD56+NK cells.” The CD56+ cells or CD3−/CD56+ cells may includecells in which CD56 glycoprotein on the cell surface is expressed, orfurther, cells in which CD3 glycoprotein is not expressed while the CD56glycoprotein is expressed. Even the same type of immune cells may havedifferences in CD type attached to the cell surface and expression rateand thus, the functions thereof may be different.

Second Isolation Step

In some embodiments, the isolating of the CD56+ natural killer cellsfrom the blood sample may be performed by an isolating method using atleast one selected from the group consisting of CD56 microbeads and CD3microbeads, or an isolating method using equipment such as CliniMACSs, aflow cytometry cell sorter, etc.

For example, the isolating method using the CD56 microbeads and/or theCD3 microbeads may be performed by adding the CD56 microbeads to PBMCsand then removing non-specific binding, or performed by adding the CD3microbeads to the PBMCs to remove specific binding and then adding theCD56 microbeads again to remove non-specific binding. In some instances,through isolating CD56+ cells and/or CD3−/CD56+ cells from PBMCs, Tcells or other non-natural killer cells may be removed.

Culturing Step

In the present specification, the term “cytokine” may refer to animmunoactive compound that is usable to induce the peripheral bloodmononuclear cells to differentiate into NK cells.

In some embodiments, the cytokine may be interleukin-2 (IL-2), IL-15,IL-21, FMS-like tyrosine kinase 3 ligand (Flt3-L), a stem cell factor(SCF), IL-7, IL-18, IL-4, type I interferons, a granulocyte-macrophagecolony-stimulating factor (GM-CSF), and an insulin-like growth factor 1(IGF 1), but not limited thereto.

In some embodiments, the cytokine may be used at a concentration of50-1,000, 50-900, 50-800, 50-700, 50-600, 50-550, 100-550, 150-550,200-550, 250-550, 300-550, 350-550, 400-550, 450-550 IU/mL. Conventionalmethods of proliferating NK cells utilize high concentrations of variouscytokines. Conversely, in some embodiments of the method ofproliferating NK cells described herein, since two types of feeder cellsmay be used with the high-purity CD56+ cells, NK cells with high yieldand high purity may be proliferated using only low concentrations of onecytokine.

In the present specification, the term “feeder cell” may refer to a cellthat does not divide and proliferate, but has metabolic activity toproduce various metabolites and thus, helps the proliferation of targetcells.

In some embodiments, the feeder cells may be at least one selected fromthe group consisting of irradiated Jurkat cells, irradiated Epstein-Barrvirus transformed lymphocyte continuous line (EBV-LCL) cells, and PBMC,HFWT, RPMI 1866, Daudi, MM-170, K562 or cells genetically modified bytargeting K562 (for example, K562-mbIL-15-41BB ligand). For example, inone embodiment, the feeder cells may be the irradiated Jurkat cells andthe EBV-LCL cells.

In the present specification, the term “Jurkat cell” or “Jurkat cellline” may refer to a blood cancer (immortalized acute T cell leukemia)cell line, which has been developed by Dr. Arthur Weiss of theUniversity of California at San Francisco. Jurkat cells, in whichvarious chemokine receptors are expressed and capable of producing IL-2,have not generally been considered as a possible candidate of the feedercells for anti-cancer immunotherapy because MHC class 1, which is anatural killer cell activation inhibitor, is highly expressed on thecell surface thereof. The Jurkat cells may be obtained from the ATCC(ATCC TIB-152).

In the present specification, the term “EBV-LCL cell” or “EBV-LCL cellline” refers to an Epstein-Barr virus transformed lymphocyte continuousline (EBV-LCL) (D. M. Koelle et al., J Clin Invest, 1993: 91: 961-968),which is a B cell line that is made by infecting human B cells withEpstein-Barr virus in a test tube. The EBV-LCL cells may be directlyprepared and used in a general laboratory by a method of addingcyclosporine A in a process of infecting EBV in the PBMC. In someembodiments, the EBV-LCL cell may be prepared by following steps. 30×10⁶PBMCs are added in 9 mL of a culture medium, the mixture is added in a T25 culture flask, and then 9 mL of an EBV supernatant is added. 80 μL ofcyclosporine A (50 μg/mL) is added and then cultured at 37° C. After 7days of culture, a half of supernatant is removed, a fresh culturemedium is added, and then 40 μL of cyclosporine A is added. The sameprocess may be repeated once every 7 days until 28 days of culture. Thecell line may be usable after 28 days of culture, and from this time,the cell line may be cultured in the culture medium without addingcyclosporine A.

The Jurkat cells and the EBV-LCL cells may be used as the feeder cellsafter irradiation.

In some embodiments, the irradiated Jurkat cells and the irradiatedEBV-LCL cells may be included at a content ratio of 1:0.1-5, 1:0.1-4,1:0.1-3, 1:0.1-2, 1:0.1-1.5, 1:0.5-1.5, 1:0.75-1.25, 0.1-5:1, 0.1-4:1,0.1-3:1, 0.1-2:1, 0.1-1.5:1, 0.5-1.5:1 or 0.75-1.25:1. For example, theirradiated Jurkat cells and the irradiated EBV-LCL cells may be includedat a content ratio of 1:1.

In some embodiments, the irradiated Jurkat cells and the irradiatedEBV-LCL cells may be obtained by treating with irradiation of 50-500,50-400, 50-300, 50-200, 50-150, 70-130, 80-120 or 90-110 Gy. Forexample, the irradiated Jurkat cells and/or the irradiated EBV-LCL cellsmay be obtained by treating Jurkat cells and/or EBV-LCL cells withirradiation of 100 Gy.

In some embodiments, the culturing may be performed for 1-50, 1-42,1-40, 1-35, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15 or 1-14 days.

In some embodiments, the culturing step may further include followingsteps: co-culturing with the feeder cells and a first cytokine (“firstculturing step”); and further co-culturing after addition of a secondcytokine (“second culturing step”)

The second culturing step may include adding the second cytokine once ormore between day 0-6 of culturing. For example, the second culturingstep may include adding the second cytokine once on each of day 0 andday 3 of culturing.

The second culturing step may include adding the second cytokine and thefeeder cells during the first 6 days of the cycle of 14 days ofculturing. For example, the second culturing step may include adding thefeeder cells during a 14 days cycle, and adding the second cytokine onday 3 and 6 of each cycle once each.

In some embodiments, the first cytokine may be IL-2. In someembodiments, the second cytokine may be IL-21. In some embodiments, thesecond cytokine may be used at the concentration of 10-1000, 10-500,10-100, 20-100, 30-100, 40-100, 50-100 or 10-50 ng/mL. In someembodiments, culturing with the addition of the second cytokine once ormore during day 0-6 may exhibit superior proliferation and/oranti-cancer activity. In some embodiments, culturing with the additionof the feeder cells and the second cytokine for six days in the cycle of14 days may exhibit superior proliferation and/or anti-cancer activity.

In some embodiments, the co-culturing may be performed by including theperipheral blood mononuclear cells and the feeder cells (for example,the Jurkat cells and the EBV-LCL cells) at a mixing ratio of 1:1-100,1:1-90, 1:1-80, 1:1-70, 1:10-65, 1:20-65, 1:30-65, 1:40-65, 1:50-65 or1:55-65.

The co-culturing may be performed in a medium and any suitable mediagenerally used for induction and proliferation of the peripheral bloodmononuclear cells to the NK cells in the art may be used without alimitation as such a medium. For example, an RPMI-1640, DMEM, x-vivo0,x-vivo20, or cellgro SCGM medium may be used as such a medium. Inaddition, the culture conditions such as a temperature may follow anysuitable culture conditions of the peripheral blood mononuclear cellsknown in the art.

In some embodiments, within the produced NK cells, a ratio or purity ofthe CD56+NK cells may be 85% or more, 90% or more, or 95% or more, or98% or more with respect to the whole cells. In some embodiments, withinthe produced NK cells, a ratio of T cells to whole cells may be 15% orless, 10% or less, 5% or less, 2% or less, 1% or less.

Cell Therapeutic Composition for Treating Cancer

According to some embodiments, a cell therapeutic composition for thetreatment of cancer may include peripheral blood derived CD56+NK cellsand a cytokine.

In the present specification, the term “peripheral blood-derived” maymean that the cells are derived from “whole blood of the peripheralblood” or “leukocytes isolated from the peripheral blood usingleukapheresis.” The peripheral blood derived CD56+NK cells may be usedinterchangeably with peripheral blood mononuclear cell (PBMC) derivedCD56+NK cells.

In some embodiments, the cytokine may be used at a concentration of18-180,000, 20-100,000, 50-50,000, 50-1,000, 50-900, 50-800, 50-700,50-600, 50-550, 100-550, 150-550, 200-550, 250-550, 300-550, 350-550,400-550, 450-550 IU/mL. When the cytokine is used in these ranges, itmay suppress apoptosis of the NK cells included in the cancer treatmentcomposition and increase anti-cancer activity of the NK cells.

In some embodiments, the composition may include IL-2 as the cytokine.

In some embodiments, the CD56+NK cells may be obtained as describedelsewhere herein. For example, the CD56+NK cells may be obtained bycoculturing with feeder cells (e.g. irradiated Jurkat cells andirradiated EBV-LCL cells). In some embodiments, the ratio of CD56+NKcells to whole cells (purity) may be 85% or more, 90% or more, 95% ormore, or 98% or more.

In some embodiments, the cancer may be blood cancer, stomach cancer,pancreatic cancer, cholangiocarcinoma, colon cancer, breast cancer,liver cancer, ovarian cancer, lung cancer, kidney cancer, prostatecancer or neuroblastoma, but not limited thereto.

In some embodiments, the composition may not include T cells, or mayinclude only trace amount of T cells. For example, the ratio of T cellsto whole cells in the composition may be less than 15%, less than 10%,less than 5%, less than 2%, less than 1% or less.

In the present specification, the term “T cell” refers to a lymphocytederived from thymus, which can “memorize” previously encounteredantigens and provide information to B cells, thereby facilitatesproduction of antibody and plays an important role in cell immunesystem. Since these T cells may distinguish very small differences amongdifferent antigens to induce an immune response to allogenic antigens,autologous therapy is possible, but there may be a limit to be used forallogenic therapy. Accordingly, the cell therapeutic composition withoutT cells may be suitable for allotransplantation.

In the present specification, the term “cell therapeutic agent” refersto a medicine which is used for treatment, diagnosis, and preventionthrough a series of actions, such as proliferating and screeningautologous, allogenic, and xenogenic living cells in vitro for restoringfunctions of cells and tissues or changing biological characteristics ofthe cells by other methods. The cell therapeutic agents have beenregulated as medical products from 1993 in USA and 2002 in Korea. Thesecell therapeutic agents may be largely classified into two fields, thatare, first, stem cell therapeutic agents for tissue regeneration orrecovery of organ functions, and second, immune cell therapeutic agentsfor regulation of immune responses, such as inhibition of the immuneresponse or enhancement of the immune response in vivo.

An administration route of cell therapeutic compositions describedherein may be any suitable route as long as the composition reaches atarget tissue. The administration may be parenteral administration, forexample, intraperitoneal administration, intravenous administration,intramuscular administration, subcutaneous administration, orintradermal administration, but not limited thereto.

The cell therapeutic composition described herein may be formulated in asuitable form together with a pharmaceutically acceptable carriersuitable or generally used for cell therapy. The “pharmaceuticallyacceptable” refers to a composition which is physiologically acceptableand does not generally cause an allergic reaction such asgastrointestinal disorders, dizziness, or the like, or similar reactionsthereto, when being administered to the human body. The pharmaceuticallyacceptable carrier may include, for example, parenteral administrationcarries such as water, suitable oils, saline, aqueous glucose andglycol, and the like, and further include stabilizers and preservatives.The suitable stabilizer includes an antioxidant such as sodium hydrogensulfite, sodium sulfite, or ascorbic acid, sucrose, albumin, or thelike. The suitable preservative includes DMSO, glycerol, ethyleneglycol, sucrose, trehalose, dextrose, polyvinylpyrrolidone, or the like.

The cell therapeutic composition may also be administered by any devicein which the cell therapeutic agent may move to the target cell.

The cell therapeutic composition may include a therapeutically effectiveamount of cell therapeutic agent for treatment of diseases. The term“therapeutically effective amount” means an amount of an activeingredient or a cell therapeutic composition which induces biological ormedical responses in tissue systems, animals, or humans which areconsidered by researchers, veterinarians, physicians, or otherclinicians, and includes an amount of inducing alleviation of symptomsof diseases or disorders to be treated. It will be apparent to thoseskilled in the art that the cell therapeutic agent included in the celltherapeutic composition may be changed according to a desired effect.Therefore, the optimal content of the cell therapeutic agent may beeasily determined by those skilled in the art, and may be adjustedaccording to various factors including a type of disease, severity ofthe disease, contents of other ingredients contained in the composition,a type of formulation, and an age, a weight, a general health condition,a gender, and a diet of a patient, an administration time, anadministration route, a secretion ratio of the composition, a treatmentperiod, and simultaneously used drugs. It is important to include anamount capable of obtaining a maximum effect by a minimum amount withoutside effects by considering all of the factors. For example, the celltherapeutic composition may include a cell therapeutic agent of 1×10⁶ to5×10⁸ cells per kg of body weight.

Method for Preventing or Treating Cancer

Further, according to another aspect of the invention, a method forpreventing or treating cancer is provided, the method comprisingadministering a cell therapeutic composition for anti-cancer includingperipheral blood-derived CD56+ natural killer cells and cytokines to asubject. The term “subject” refers to a mammal which is a subject fortreatment, observation, or testing, and preferably, a human. The subjectmay be a patient of blood cancer, stomach cancer, pancreatic cancer,cholangiocarcinoma, colon cancer, breast cancer, liver cancer, ovariancancer, lung cancer, kidney cancer, prostate cancer or neuroblastoma,but not limited thereto.

In some embodiments, in the case of an adult, the cell therapeuticcomposition may be administered once to several times a day. The celltherapeutic composition may be administered every day or in a 2-180 dayinterval. the cell therapeutic agent included in the composition mayinclude 1×10⁶ to 1×10¹¹ peripheral blood-derived CD56+ natural killercells, for example, about 1×10⁶ to 1×10⁹ NK cells per kg of body weight.In some embodiments, the peripheral blood-derived CD56+ natural killercells in the cell therapeutic composition are at least about 90% pure.In some embodiments, the cytokine is IL-2 at a concentration rangingfrom about 50-50,000 IU/ml.

In some embodiments, the cell therapeutic composition of the presentinvention may be administered by any suitable method, such asadministration through a rectal, intravenous, intraarterial,intraperitoneal, intramuscular, intrasternal, percutaneous, topical,intraocular, or intradermal route. In some embodiments, the NK cellsincluded in the composition may be allogenic, i.e. obtained from aperson other than the subject being treated. In some embodiments, theperson may be a normal person or a cancer patient. In some embodiments,the NK cells included in the composition may be autologous, i.e.obtained from the subject being treated.

In some embodiments, the NK cells disclosed herein and the celltherapeutic composition including the NK cells disclosed herein may beused for treating disease or condition other than cancer. It has beenreported that NK cells plays an important role in the regulation ofimmune system, for example, by regulating of T-cells, thus the celltherapeutic composition having the NK cells may be administered to treatconditions associated with the immune system. For example, the celltherapeutic composition may be administered to treat neurodegenerativedisorders (e.g. Alzheimer's disease and Parkinson's disease) orautoimmune diseases (e.g. rheumatoid arthritis, multiple sclerosis,psoriasis, spondyloarthropathies, SLE, Sjogren's syndrome, systemicsclerosis).

Advantageous Effects

Features and advantages of the present invention are summarized asfollows:

(a) The present invention relates to a method of producing naturalkiller cells.

(b) According to the method of producing natural killer cells, since thehigh-purity natural killer cells in which the T cells and the like areremoved can be produced without using various expensive cytokines, it ispossible to enhance an effect of prevention and treatment of cancer,particularly, allogenic therapy using the natural killer cells.

(c) The present invention relates to a cell therapeutic composition foranti-cancer comprising peripheral blood-derived CD56+NK cells andcytokines.

(d) The composition of the present invention includes high-puritynatural killer cells with minimal (e.g., less than about 1%) T cells,and thus the composition may be effectively used for allogenic therapyas well as autologous therapy.

Examples

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the disclosure to the particular features or embodimentsdescribed.

Example 1. Production of CD56+ Natural Killer (NK) Cells

CD56+ cells and CD3−/CD56+ cells were isolated from PBMCs by thefollowing method. First, the PBMCs were isolated from the blood using aFicoll-Hypaque density gradient method and then the cells were counted.

Example 1-1. Preparation for Producing CD56+ Cells

The counted PBMCs were added with a MACS buffer (1×PBS+0.5% HSA) andsuspended, and added with CD56 microbeads (Miltenyi Biotec) to be 1 to20 μL per 1.0×10⁷ PBMCs, and then incubated at 2 to 8° C. for 5 to 30minutes. After incubation, the MACS buffer was added and mixed, and thenthe mixture was centrifuged (600×g) to precipitate the cells. Aftercentrifugation, a supernatant was removed, and the cells were suspendedby adding the MACS buffer and added in a column connected to a MACSseparator. The MACS buffer passed through the column to removenon-specific binding. The column was separated from the MACS separatorand transferred to a 15 mL conical tube, and then added with the MACSbuffer to isolate CD56+ cells attached to the column.

Example 1-2. Preparation for Producing CD3−/CD56+ Cells

The counted PBMCs were added with a MACS buffer (1×PBS±0.5% HSA) andsuspended, and added with CD3 microbeads (Miltenyi Biotec) to be 1 to 20μL per 1.0×10⁷ PBMCs, and then incubated at 2 to 8° C. for 5 to 30minutes. After incubation, the MACS buffer was added and mixed, and thenthe mixture was centrifuged (600×g) to precipitate the cells. Aftercentrifugation, a supernatant was removed, and the cells were suspendedby adding the MACS buffer and added in a column connected to a MACSseparator. The MACS buffer passed through the column to collect CD3−cells. The collected CD3− cells were added with a MACS buffer(1×PBS+0.5% HSA) and suspended, and added with CD56 microbeads (MiltenyiBiotec) to be 1 to 20 μL per 1.0×10⁷ CD3− cells, and then incubated at 2to 8° C. for 5 to 30 minutes. After incubation, the MACS buffer wasadded and mixed, and then the mixture was centrifuged (600×g) toprecipitate the cells. After centrifugation, a supernatant was removed,and the cells were suspended by adding the MACS buffer and added in acolumn connected to a MACS separator. The MACS buffer passed through thecolumn to remove non-specific binding. The column was separated from theMACS separator and transferred to a 15 mL conical tube, and then addedwith the MACS buffer to isolate CD3−/CD56+ cells attached to the column.

Example 1-3. Production of NK Cells Using the CD56+ Cells and CD3−/CD56+Cells

The CD56+ cells or the CD3−/CD56+ cells isolated from the PBMCs as inExamples 1-1 and 1-2 were added in a RPMI-1640 medium containing FBS 10%added with IL-2 at a concentration of 500 IU/mL together with preparedcombination of feeder cells (Jurkat cells and EBV-LCL cells) irradiatedwith 100 Gy radiation and then co-cultured in an incubator at 37° C. and5% CO₂. The ratio of (CD56+ cells and/or CD3−/CD56+ cells):(Jurkatcells):(EBV-LCL cells) was about 1:30:30.

Meanwhile, the Jurkat cells may be obtained from ATCC (ATCC TIB-152),and the EBV-LCL cells were prepared by the following method: 30×10⁶PBMCs were added in 9 mL of a culture medium, the mixture was added in aT 25 culture flask, and then 9 m of an EBV supernatant was added. 80 μLof cyclosporine A was added and then cultured at 37° C. After 7 days ofculture, a half of supernatant was removed, a fresh culture medium wasadded, and then 40 μL of cyclosporine A was added. The same process asthe 7th day was repeated once every 7 days until 28 days of culture. Thecell line was usable after 28 days of culture, and from this time, thecell line was cultured in the culture medium without adding cyclosporineA.

Example 2. Production of CD56+ Natural Killer (NK) Cells (IL-2/IL-21Treated)

NK cells were produced using same method of Example 1 (1-1 to 1-3),except for adding IL-2 (500 IU/mL) and IL-21 (50 ng/mL) instead of IL-2(500 U/mL).

Comparative Example 1. Production of Natural Killer (NK) Cells withoutthe CD56+ Cells Isolation Step (IL-2 Treated)

PBMCs were isolated from the blood using a Ficoll-Hypaque densitygradient method. The PBMCs were added in a RPMI-1640 medium containingFBS 10% added with IL-2 at a concentration of 500 IU/mL together withprepared feeder cells (Jurkat cells and EBV-LCL cells) irradiated with100 Gy radiation and then co-cultured in an incubator at 37° C. and 5%CO₂.

Comparative Example 2. Production of Natural Killer (NK) Cells withoutthe CD56+ Cells Isolation Step (IL-2/IL-21 Treated)

NK cells were produced using same method of Comparative Example 1,except for adding IL-2 (500 IU/mL) and IL-21 (50 ng/mL) instead of IL-2(500 IU/mL).

Comparative Examples 3&4. Production of Natural Killer (NK) Cellswithout the CD56+ Cells Isolation Step

NK cells were produced using similar methods of Comparative Examples1&2, respectively, except for that a ratio of PBMC:(Jurkatcells):(EBV-LCL cells) was 1:0.5:0.5.

Experimental Example 1. Confirmation of Proliferation Ability of NKCells

With respect to each of the NK cells cultured in a CO₂ incubatoraccording to Examples 1, 2 and Comparative Examples 1, 2, on Day 6 ofculture in a T 25 culture flask, cells were inoculated into a 350 mL bagon the basis of the cell number of 1.0×10⁵ to 2.0×10⁶/mL and furthercultured for 4 days. On Day 10 of culture, the cells were inoculatedinto a 1 L bag on the basis of the cell number of 1.0×10⁵ to 2.0×10⁶/mLand then further cultured for 4 days. Finally, on Day 14 of culture, thecells were inoculated into a 1 L bag on the basis of the cell number of1.0×10⁵ to 2.0×10⁶/mL and then further cultured for 3 to 6 days.

FIG. 1A illustrates the fold increase of NK cells during the culture. Asillustrated in FIG. 1A and Table 1 below, the CD56+NK cells (CD56+ andCD3−/CD56+) of Example 1 were proliferated 2675 and 1903 timesrespectively on Day 17 compared to Day 0, while the PBMC cells ofComparative Example 1 was proliferated 1768 times on Day 17 compared toDay 0.

TABLE 1 Expansion Folds DAY 0 DAY 6 DAY 10 DAY 14 DAY 17 PBMC 1 2 52 6081768 CD56+ 1 8 188 1311 2675 CD3−/CD56+ 1 6 142 966 1903

FIG. 1B illustrates the fold increase and the population doubling level(PDL) of NK cells. Further, as illustrated in FIG. 1B, the PBMCs ofComparative Example 1 (PBMC w/o IL-21) and Comparative Example 2 (PBMCw/IL-21) were proliferated 243 and 1248 times respectively compared toDay 0, while the CD56+NK cells of Example 1 (CD56 w/o IL-21) and Example2 (CD56 w/IL-21) were proliferated 2990 and 20434 times respectivelycompared to Day 0.

Experimental Example 2. Confirmation of Purity of CD56+NK Cells

The NK cells of Examples 1, 2 and Comparative Examples 1, 2 were washedonce with a FACS staining buffer and suspended in 100 μL, and thenstored at 2 to 8° C. for 20 to 30 minutes under a dark condition aftermixing with a monoclonal antibody binding with fluorescence. After oneadditional washing, the cells were suspended in 300 to 500 μL of theFACS staining buffer and then 10,000 to 100,000 cells per tube wereobtained and analyzed by using a CD56-FITC/CD3-PE/CD20-PerCP5/CD14-APCpanel of a flow cytometer. The purity of the CD56+NK cells was definedas a ratio of cells introduced in a CD3−/CD56+ region after FSC/SSCgating, and it was further confirmed that CD20 and CD14 were notexpressed in the cells in the CD3−/CD56+ region.

As illustrated in FIG. 2, the purity of NK cells of Comparative Example1 (PBMCs w/o IL-21) and Comparative Example 2 (PBMCs w/IL-21) were 84.2%and 84.7% respectively, while the purity of NK cells of Example 1 (CD56w/o IL-21) and Example 2 (CD56 w/IL-21) were 98.6% and 99.2%respectively.

Experimental Example 3. Confirmation of Cancer Cell Cytotoxicity of NKCells

First, the cytotoxicity against K562 cells (blood cancer, ATCC®CCL-243™), a chronic myelogenous leukemia cell line was confirmed.

Before used in the experiment, K562 cells were prepared by subculturingK562 cells suspended in a RPMI 1640 medium containing FBS 10%, at 37±1°C. at an interval of three days, for 7 days or more.

The prepared K562 cells were suspended in the RPMI-1640 medium at aconcentration of 1.0×10⁶ cells/mL, and added with a fluorescent material(Calcein-AM) at a concentration of 4 μM. The K562 cells were stained at37±1° C. for 30 minutes, and then inverted at an interval of tenminutes. The K562 cells stained with the fluorescent material werecentrifuged at 3,300 rpm for 3 minutes, washed three times, and thensuspended in an SNK medium containing FBS 10%, at a ratio of 1.0×10⁶cells/mL. The K562 cells were inoculated into a round bottom microwellplate (96-well) in an amount of 1.0×10⁴ cells per well.

The NK cells of the Experimental Example 1 (effector cells) on the 14 to20th days of culture were suspended and diluted in a RPMI-1640 mediumcontaining FBS 10% at ratios of 1.0×10⁶ cells/mL, 3.0×10⁵ cells/mL,1.0×10⁵ cells/mL and 0.5×10⁵ cells/mL, respectively.

The diluted effector cells were inoculated into the plate inoculatedwith the target cells (the K562 cells) at a concentration of 100 μL perwell for three wells each (triplication), respectively. In this case,ratios of the effector cells and the target cells are shown in Table 2below.

TABLE 2 Effector:Target Effector cells Target cells 10:1  1.0 × 10⁵ 1.0× 10⁴ 3:1 3.0 × 10⁴ 1.0 × 10⁴ 1:1 1.0 × 10⁴ 1.0 × 10⁴ 0.5:1  0.5 × 10⁴1.0 × 10⁴

The plate design used in the present experiment is shown in FIG. 3, in anegative control group (Spontaneous), fluorescence-stained living K562cells were added, and in a positive control group (Maximum release), theK562 cells were completely killed using TX-100 and exhibited a maximumfluorescence.

The plate inoculated with the target cells and the effector cells wascentrifuged at 1000 rpm for 5 minutes, cultured at 37±1° C. for 3 to 4hours, and then centrifuged again at 1,000 rpm for 5 minutes. Aftercentrifugation, 80 μL of a supernatant was transferred to a black plate(96-well), and then a fluorescence amount was measured using afluorescence microplate reader and the cytotoxicity against cancer cellswas calculated using Equation 1 below.

$\begin{matrix}{{Cytotoxicity} = {\frac{{{Test}\mspace{14mu} {Release}} - {{Spontaneous}\mspace{14mu} {Release}}}{{{Maximum}\mspace{14mu} {Release}} - {{Sponteneous}\mspace{14mu} {Release}}} \times 100}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

FIG. 4A and Table 3 below show % of lysis of K562 cells at various E:Tratio. As illustrated in FIG. 4A and Table 3 below, as compared withComparative Example 3 (PBMCs w/o IL-21) and Comparative Example 4 (PBMCsw/IL-21), the CD56+ cells cultured according to Example 1 (CD56+w/oIL-21) and Example 2 (CD56+w/IL-21) exhibited higher anti-canceractivity.

TABLE 3 % of lysis E:T (10:1) E:T (3:1) E:T (1:1) E:T (0.5:1) PBMC(W/OIL-21) 97.5 94.6 75.3 60.0 PBMC(W/IL-21) 102.3 97.1 84.8 66.9 CD56+(W/OIL-21) 103.3 99.9 83.8 67.4 CD56+(W/IL-21) 102.9 100.7 87.7 80.0

Next, the cytotoxicity against solid tumor cells, which are known tohave greater tolerance against NK cells, is confirmed. AGS (stomachcancer, ATCC® CRL-1739™), A549 (lung cancer, ATCC® CRL-185™), andMDA-MB0231 (breast cancer, ATCC® HTB-26™) were used as solid tumor celllines.

Each solid tumor cells were tagged with green-fluorescent marker usingCYTO-ID® Green long-term tracer kit (Enzo Life Sciences Inc.),inoculated on a plate, and cultured for 24 hours. Next day, NK cells andcancer cells were reacted for 48 hours in 0.5:1 ratio. After 48 hours,cytotoxicity was confirmed by measuring the number of cells exhibitinggreen-fluorescence using flow cytometer.

As illustrated in FIG. 4B, as compared with Comparative Example 1 (PBMCsw/o IL-21) and Comparative Example 2 (PBMCs w/IL-21), the CD56+ cellscultured according to Example 1 (CD56+w/o IL-21) and Example 2(CD56+w/IL-21) exhibited higher anti-cancer activity.

Experimental Example 4. Comparison of Proliferative Ability of NK CellsDepending on Timing and Number of IL-21 Treatment

To evaluate the proliferative ability of NK cells according to thetiming of IL-21 treatment, experiments as outlined below were conducted.

CD56+NK cells were produced according to the method of Example 1, buttreated with IL-21 (50 ng/mL) during Day 0-6 (D0-6 group), Day 6-10(D6-10 group), Day 10-14 (D10-14 group), or Day 14-17 (D-14-17 group),and the proliferative ability of the CD56+NK cells were compared usingthe method according to Experimental Example 1.

NK cells were treated with IL-21: for the D0-6 group, twice, on Day 0and 3; for the D6-10 group, once, on Day 6; for the D10-14 group, once,on Day 10; for the D14-17 group, once, on Day 14. For a control group,NK cells were not treated with IL-21.

As shown in FIG. 5A and Table 4, the D10-14 group and the D14-17 groupdid not exhibit significant difference in proliferative ability ascompared with the control group, while the D0-6 group and the D6-10group exhibited increased proliferation ability as compared with thecontrol group. Especially, the D0-6 group exhibited the greatestexpansion fold increase.

TABLE 4 Expansion Folds control D 0-6 D 6-10 D 10-14 D 14-17 Donor 12996 21859 6388 2894 2330

To evaluate the proliferative ability of NK cells according to thenumber of IL-21 treatments, experiments as outlined below wereconducted.

CD56+NK cells were produced according to the method of Example 1, buttreated with IL-21 (50 ng/mL) during Day 0-3 (D0-3 group), Day 3-6 (D3-6group), or Day 0-6 (D0-6 group), and the proliferative ability of theCD56+NK cells were compared using the method according to ExperimentalExample 1.

NK cells were treated with IL-21: for the D0-3 group, once, on Day 0;for the D3-6 group, once, on Day 3; for the D0-6 group, twice, on Day 0and 3. For a control group, NK cells were not treated with IL-21.

As shown in FIG. 5B and Table 5, every group with IL-21 treatment duringearlier stage of the culture exhibited increased expansion fold ascompared with the control group. Especially, D0-6 exhibited the greatestexpansion fold increase.

TABLE 5 Expansion Fold control D 0-3 D 3-6 D 0-6 Donor 1 2996 16420 436021859

Experimental Example 5. Comparison of Proliferative Ability of NK CellsDepending on the Concentration of IL-21 Treatment

CD56+NK cells were produced according to the method of Example 1, buttreated with IL-21 with a concentration of 0 ng/mL, 10 ng/mL, 30 ng/mL,50 ng/mL or 100 ng/mL twice, and the proliferative ability of theCD56+NK cells were compared using the method according to ExperimentalExample 1.

As shown in FIG. 6, even when treated with IL-21 with a concentration of10 ng/mL, the NK cells exhibited greater expansion fold as compared withthe NK cells with no IL-21 treatment, and the expansion fold of the NKcells increased as the concentration of IL-21 increases between 10ng/mL-50 ng/mL. However, when treated with IL-21 with a concentration of100 ng/mL, the NK cells did not exhibit significant difference inexpansion from the NK cells treated with IL-21 with a concentration of50 ng/mL.

Experimental Example 6. Comparison of Cytotoxicity of NK Cells Dependingon the Timing and Number of IL-21 Treatment

To evaluate the cytotoxicity of NK cells against cancer cells accordingto the timing of IL-21 treatment, experiments as outlined below wereconducted.

CD56+NK cells were produced according to the method of Example 1, buttreated with IL-21 (50 ng/mL) during Day 0-6 (D0-6 group), Day 6-10(D6-10 group), Day 10-14 (D10-14 group), or Day 14-17 (D-14-17 group),and the cytotoxicity of the CD56+NK cells against blood cancer cells(K562 cells, CCL-243™) were compared using the method according toExperimental Example 3.

NK cells were treated with IL-21: for the D0-6 group, twice, on Day 0and 3; for the D6-10 group, once, on Day 6; for the D10-14 group, once,on Day 10; for the D14-17 group, once, on Day 14. For a control group,NK cells were not treated with IL-21.

As shown in FIG. 7A and Table 6, all groups of NK cells with IL-21treatment, except the D14-17 group, exhibited greater anti-canceractivity as compared with the control group.

TABLE 6 E:T ratio 10:1 3:1 1:1 0.5:1 Control (No treat) 98.8 96.9 73.155.1 D 0-6 98.6 97.0 77.8 68.4 D 6-10 96.78 99.1 80.3 69.8 D 10-14 98.496.6 68.5 52.4 D 14-17 104.5 98.8 79.1 68.8

Further, for each of the produced groups of CD56+NK cells, thecytotoxicity of the CD56+NK cells against solid tumor cells werecompared using the method according to Experimental Example 3. AGS(stomach cancer, ATCC® CRL-1739™), A549 (lung cancer, ATCC® CRL-185™),and MDA-MB0231 (breast cancer, ATCC® HTB-26™) were used as solid tumorcell lines.

As shown in FIG. 7B, the NK cells with IL-21 treatment during an earlierstage of the culture (the D0-6 group) exhibited the greatest anti-canceractivity against all three types of solid tumor cells.

To evaluate the cytotoxicity of the NK cells according to the number ofIL-21 treatments, experiments as outlined below were conducted.

CD56+NK cells were produced according to the method of Example 1, buttreated with IL-21 (50 ng/mL) during Day 0-3 (D0-3 group), Day 3-6 (D3-6group), or Day 0-6 (D0-6 group), and the cytotoxicity of the CD56+NKcells against solid tumor cells were compared using the method accordingto Experimental Example 3. AGS (stomach cancer, ATCC® CRL-1739™), A549(lung cancer, ATCC® CRL-185™), and MDA-MB0231 (breast cancer, ATCC®HTB-26™) were used as solid tumor cell lines.

NK cells were treated with IL-21: for the D0-3 group, once, on Day 0;for the D3-6 group, once, on Day 3; for the D0-6 group, twice, on Day 0and 3. For a control group, NK cells were not treated with IL-21.

As shown in FIG. 7C, every group with IL-21 treatment during earlierstages of the culture exhibited greater anti-cancer activity as comparedwith the control group.

Experimental Example 7. Comparison of Cytotoxicity of NK Cells Dependingon the Concentration of IL-21 Treatment

CD56+NK cells were produced according to the method of Example 1, buttreated with IL-21 with a concentration of 0 ng/mL, 10 ng/mL, 30 ng/mL,50 ng/mL or 100 ng/mL twice, and the cytotoxicity of the CD56+NK cellsagainst blood cancer cells (K562 cells, CCL-243™) were compared usingthe method according to Experimental Example 3.

As shown in FIG. 8A, most NK cells treated with IL-21 exhibited greatercytotoxicity as compared with the NK cells with no IL-21 treatment, whentreated with IL-21 with a concentration of 100 ng/mL, the NK cells didnot exhibit significant difference in expansion from the NK cells nottreated with IL-21.

CD56+NK cells were produced according to the method of Example 1, buttreated with IL-21 with a concentration of 0 ng/mL, 10 ng/mL, 30 ng/mL,50 ng/mL or 100 ng/mL twice, and the cytotoxicity of the CD56+NK cellsagainst solid tumor cells (K562 cells, CCL-243™) were compared using themethod according to Experimental Example 3. AGS (stomach cancer, ATCC®CRL-1739™), A549 (lung cancer, ATCC® CRL-185™), and MDA-MB0231 (breastcancer, ATCC® HTB-26™) were used as solid tumor cell lines.

As shown in FIG. 8B, the NK cells treated with IL-21 with aconcentration of 50 ng/mL exhibited the greatest anti-cancer activity.

Experimental Example 8. Comparison of Proliferative Activity of NK CellsDepending on the Number of Feeder Cell Treatment

To analyze whether multiple treatments with feeder cells would sustainproliferation of NK cells, the NK cells during the culture were treatedwith feeder cells in an interval of 14 days, and the expansion of NKcells were monitored for 42 days.

To further analyze the increase of NK cells expansion depending on IL-21treatment, the NK cells were treated with IL-21 (50 ng/mL) twice in 3days interval, during a six days period from each treatment with feedercells (Day 0-6, 14-20, 28-34).

As shown in FIG. 9, when treated with feeder cells twice or more andIL-21 together, the NK cells exhibited significantly increased expansionfold, and the NK cells treated with IL-21 exhibited greater expansionfold on Day 42, as compared with the NK cells not treated with IL-21(approximately 3.4×10¹⁰ vs. approximately 5.3×10⁸).

Experimental Example 9. Confirmation of the Effect of Culturing of NKCells Using Blood of Certain Cancer Patients

CD56+NK cells were produced according to the method of Example 1 for 17days, except that PBMCs of colorectal cancer patients was used. Theproliferative ability and the purity of the produced NK cells wasmeasured using methods according to Experimental Examples 1 and 2.

For some groups, the NK cells were treated with IL-21 with aconcentration of 50 ng/mL twice (Day 0 and Day 3 of culture), to confirmthe effect of IL-21 treatment.

As illustrated in FIG. 10A, the number of the NK cells not treated withIL-21 increased 8 times from Day 0, while when treated with IL-21, thenumber of the NK cells increased 1461 times from Day 0.

Further, as illustrated in FIG. 10B, the purity of the NK cells nottreated with IL-21 was only 84.2%, while when treated with IL-21, thepurity of the NK cells was 99.19%.

Further, the cytotoxicity of the NK cells treated with IL-21, and NKcells not treated with IL-21 against blood cancer cells (K562 cells,CCL-243™) were compared using the method according to ExperimentalExample 3. As illustrated in FIG. 10C, the NK cells treated with IL-21exhibited greater anti-cancer activity as compared with the NK cells nottreated with IL-21.

Also, Further, for each of the NK cells treated with IL-21, and NK cellsnot treated with IL-21, the cytotoxicity of the NK cells against solidtumor cells were compared using the method according to ExperimentalExample 3. AGS (stomach cancer, ATCC® CRL-1739™), A549 (lung cancer,ATCC® CRL-185™), and MDA-MB-231 (breast cancer, ATCC® HTB-26™) were usedas solid tumor cell lines. As illustrated in FIG. 10D, the NK cellstreated with IL-21 exhibited greater anti-cancer activity as comparedwith the NK cells not treated with IL-21.

Accordingly, by using methods as set forth herein, it may be possible toproduce NK cells even for certain cancer patients who do not usuallyshow an enough growth of NK cells.

Experimental Example 10. Confirmation of the Survival Rate of NK Cellsin Therapeutic Composition

With respect to each of the NK cells cultured in a CO₂ incubatoraccording to Examples 1, 2 on Day 6 of culture, cells were inoculatedinto a 350 mL bag on the basis of the cell number of 1.0×10⁵ to2.0×10⁶/mL and further cultured for 4 days. On Day 10 of culture, thecells were inoculated into a 1 L bag on the basis of the cell number of1.0×10⁵ to 2.0×10⁶/mL and then further cultured for 4 days. Finally, onDay 14 of culture, the cells were inoculated into a 1 L bag on the basisof the cell number of 1.0×10⁵ to 2.0×10⁶/mL and then further culturedfor 3 to 6 days.

The CD56+NK cells on the 14th to 20th days of culture were washed threetimes and then suspended in a base compound (physiological saline andHartman's solution) containing 1% albumin to be 2×10⁷/mL. The cells werestored at 4° C. for 48 hours and then the cell survival rate wasmeasured.

Further, in order to compare the effect of IL-2, the CD56+NK cells werewashed and suspended in a base compound containing 1% albumin(physiological saline, and Hartmann's solution or phosphate bufferedsaline), and then added with IL-2 at a concentration of 500 IU/mL Afterbeing kept in 4° C. for 48 hours, cell survival rate was measured.

100 μL of each composition was taken to obtain a total of 2×10⁶ CD56+NKcells, washed once with 1 mL of the FACS staining buffer, centrifugedand suspended in 100 μL of an annexin V binding buffer. 5 μL of AnnexinV-FTC and 5 μL of 7-AAD (Biolegend) were added in the suspension andmixed well, stored in a dark condition, and reacted at room temperaturefor 15 minutes, and then added with 400 μL of an Annexin V bindingbuffer before flow cytometry and mixed for 5 seconds. Thereafter, 10,000to 100,000 cells per tube were obtained and analyzed. A cut-off wasdetermined by setting a test tube which was not stained with the AnnexinV-FITC and the 7-AAD as a negative control, and the survival rate wasrepresented by a percentage of fraction of cells in which the AnnexinV-FITC or the 7-AAD was negative.

As illustrated in FIG. 11, when treated IL-2 (W/IL2), the apoptosis ofthe CD56+NK cells was inhibited.

Experimental Example 11. Confirmation of the Cytotoxicity of NK Cells inTherapeutic Composition

With respect to each of the NK cells cultured in a CO₂ incubatoraccording to Examples 1, 2 on Day 6 of culture, cells were inoculatedinto a 350 mL bag on the basis of the cell number of 1.0×10⁵ to2.0×10⁶/mL and further cultured for 4 days. On Day 10 of culture, thecells were inoculated into a 1 L bag on the basis of the cell number of1.0×10⁵ to 2.0×10⁶/mL and then further cultured for 4 days. Finally, onDay 14 of culture, the cells were inoculated into a 1 L bag on the basisof the cell number of 1.0×10⁵ to 2.0×10⁶/mL and then further culturedfor 3 to 6 days.

Before used in the experiment, the cancer cell lines were prepared bysuspending under the following conditions, and sub-culturing at 37±1° C.at an interval of three days for 1 week or more:

CCRF-SB (blood cancer, ATCC® CCL-120™), AGS (stomach cancer, ATCC®CRL-1739™) and MIA-PACA2 (pancreatic cancer, ATCC® CRL-1420™): RPMImedium+10% FBS,

SNU245 (cholangiocarcinoma, KCLB No. 00245), HCT15 (colon cancer, ATCC®CCL-225™) and NIH:OVCAR-3 (ovarian cancer, ATCC® HTB-161™): RPMImedium+10% FBS+25 mM HEPES, and

MDA-MB-231 (breast cancer, ATCC® HTB-26™): DMEM medium+10% FBS.

The cancer cell lines (except for a blood cancer cell line) duringculturing were detached from a culture dish using trypsin and suspendedin the medium to be 5×10⁴/mL, and then inoculated into a 24-well plateby 1 mL per well and attached for one day. To distinguish from the NKcells, the blood cancer cell line, which was a suspended culture cell,was labeled with green fluorescence, suspended in the medium to be5×10⁴/ml, and inoculated into a 24-well plate by 1 mL per well.

First, in a 24-well plate inoculated with AGS (stomach cancer, ATCC®CRL-1739™), MIA-PACA2 (pancreatic cancer, ATCC® CRL-1420™), SNU245(cholangiocarcinoma, KCLB No. 00245), HCT15 (colon cancer, ATCC®CCL-225™) and MDA-MB-231 (breast cancer, ATCC® HTB-26™) among the cancercell lines, the CD56+NK cells were added after one day to observe thecytotoxicity, and ratios of effector cells (CD56+NK cells) and targetcells (cancer cell lines) are shown in Table 7 below.

TABLE 7 Effector:Target Effector cell Target cell 0:1  0 5 × 10⁴ 1:1  5× 10⁴ 5 × 10⁴ 1:10 5 × 10³ 5 × 10⁴ 1:20 2.5 × 10³  5 × 10⁴

The plate inoculated with the effector cells and the target cells wascultured at 37±1° C. for 1 to 3 days, and at this time, in order toobserve whether anti-cancer activity is increased by IL-2, 500 IU/ml ofIL-2 was further added to an experimental group. In a negative controlgroup, the CD56+NK cells (effector cells) were not added and there wasno anti-cancer activity reaction.

After 1 to 3 days of culture, the cells were washed with RPMI threetimes to remove the CD56+NK cells present in the suspended form, andthen the cancer cell lines remaining in the wells were detached usingtrypsin, stained with trypan blue and counted. Subsequently, the plateinoculated with the target cells and the effector cells was cultured at37±1° C. for 1 to 3 days, and then the cells labeled with greenfluorescence present in the 24 wells were counted using a flowcytometer.

The cytotoxicity for the cancer cell line was calculated using Equation2 below.

$\begin{matrix}{{Cytotoxicity} = {\frac{\begin{matrix}{{{{avg}.\mspace{14mu} {number}}\mspace{14mu} {of}\mspace{14mu} {fluorescnece}} +} \\{{cells}\mspace{14mu} {in}\mspace{14mu} {wells}\mspace{14mu} {with}\mspace{14mu} {NK}\mspace{14mu} {cells}}\end{matrix}}{\begin{matrix}{{{{avg}.\mspace{14mu} {number}}\mspace{14mu} {of}\mspace{14mu} {fluorescence}} +} \\{{cells}\mspace{14mu} {in}\mspace{14mu} {well}\mspace{14mu} {with}\mspace{14mu} {target}\mspace{14mu} {cells}\mspace{14mu} {only}}\end{matrix}} \times 100}} & {{Equation}\mspace{14mu} 2}\end{matrix}$

As a result, as illustrated in FIG. 12, it was confirmed that the cancercell cytotoxicity was increased when IL-2 was treated together (W/IL2),as compared to when only the CD56+NK cells were treated (W/O IL2).

Next, the CD56+NK cells were added in the 24-well plate to whichNIH:OVCAR-3 (ovarian cancer, ATCC® HTB-161™) cells was attached amongthe cancer cell lines to observe the cytotoxicity, and ratios of targetcells (cancer cell lines) and effector cells (CD56+NK cells) were shownin Table 8 below.

TABLE 8 Effector:Target Effector cells Target cells 0:1 0 5 × 10⁴ 1:1 5× 10⁴ 5 × 10⁴ 0.1:1  5 × 10³ 5 × 10⁴ 0.05:1   2.5 × 10³  5 × 10⁴

The plate inoculated with the effector cells and the target cells wascultured at 37±1° C. for 1 days, and, in order to observe whetheranti-cancer activity is increased by IL-2, 500 IU/ml of IL-2 was furtheradded to an experimental group. In a negative control group, the CD56+NKcells were not added and there was no anti-cancer activity reaction.

After 1 day of culture, the cells were washed with RPMI three times toremove the CD56+NK cells present in the suspended form, and then thecancer cell lines remaining in the wells were photographed using acamera.

As a result, as illustrated in FIG. 13, the cancer cell cytotoxicity wasincreased when cancer cell lines were treated together with IL-2(+112),as compared to when cancer cell lines were treated with only the CD56+NKcells(−IL2).

Next, the CD56+NK cells were added in the 24-well plate to which AGS(stomach cancer, ATCC® CRL-1739™) cells was attached among the cancercell lines to observe the cytotoxicity, and ratios of target cells(cancer cell lines) and effector cells (CD56+NK cells) were shown inTable 9 below.

TABLE 9 Effector:Target Effector cells Target cells 0:1 0 5 × 10⁴ 1:1 5× 10⁴ 5 × 10⁴ 0.1:1  5 × 10³ 5 × 10⁴ 0.05:1   2.5 × 10³  5 × 10⁴

The plate inoculated with the effector cells and the target cells wascultured at 37±1° C. for 1 days, and, in order to observe whetheranti-cancer activity is increased by IL-2, 500 IU/ml of IL-2 was furtheradded to an experimental group. In a negative control group, the CD56+NKcells were not added and there was no anti-cancer activity reaction.

After 1 day of culture, the cells were washed with RPMI three times toremove the CD56+NK cells present in the suspended form, and then thecancer cell lines remaining in the wells were photographed using acamera.

As a result, as illustrated in FIG. 14, the cancer cell cytotoxicity wasincreased when cancer cell lines were treated together with IL-2 (+IL2),as compared to when cancer cell lines were treated with only the CD56+NKcells (−IL2).

Experimental Example 12. Confirmation of Anticancer Effect of NK Cellsin Animal Models

CD56+NK cells are produced according to the method of Examples 1, 2 andComparative Examples 1, 2 for 17 days, except that PBMCs of colorectalcancer patients is used. With respect to each of the NK cells culturedin a CO₂ incubator according to Examples 1, 2 and Comparative Examples1, 2, on Day 6 of culture in a T 25 culture flask, cells are inoculatedinto a 350 mL bag on the basis of the cell number of 1.0×10⁵ to2.0×10⁶/mL and further cultured for 4 days. On Day 10 of culture, thecells are inoculated into a 1 L bag on the basis of the cell number of1.0×10⁵ to 2.0×10⁶/mL and then further cultured for 4 days. Finally, onDay 14 of culture, the cells are inoculated into a 1 L bag on the basisof the cell number of 1.0×10⁵ to 2.0×10⁶/mL and then further culturedfor 3 to 6 days.

Animal models of human cancer are constructed by xenograft of humancancer cell line into mice. Following human cancer cell lines are used:AGS (stomach cancer), MIA-PACA2 (pancreatic cancer), SNU245(cholangiocarcinoma), HCT15 (colon cancer) and NIH:OVCAR-3 (ovariancancer), and MDA-MB-231 (breast cancer). After xenograft of cancer, themice are grouped randomly and marked. The control group is injected 200L of Hartmann's solution into the vein of tail. The NK cell-treated(+IL-2) group is injected five times with 1×10⁷ NK cells/200 μL and 500IU/mL of IL-2 at 2-3-day intervals from 1 week after xenograft ofcancers into the vein of tail. The NK cell-treated (−IL-2) group isinjected five times with 1×10⁷ NK cells/200 μL at 2-3-day intervals from1 week after xenograft of cancers into the vein of tail.

To follow up tumor growth, during the study period, mice are tested forbody weight and tumor volume three time a week. Length of major axis andminor axis are measured using a caliper and tumor volume is determinedaccording to the following equation (Equation 3).

Tumor Volume (mm³)=(length of major axis (mm))×(length of minor axis(mm))²×0.5  Equation 3

The NK cell-treated (−IL-2) group exhibits a reduction in tumor growthafter 8 weeks treatment of approximately 50% compared to the controlgroup based on luciferase images for each cancer type. The NKcell-treated (+IL-2) group exhibits a further reduction in tumor growthof approximately 60% compared to the control group for each cancer type.

Experimental Example 13. Confirmation of Anticancer Effect of NK Cellsin Cancer Patients

CD56+NK cells are produced according to the method of Examples 1, 2 andComparative Examples 1, 2 for 18 days, except that PBMCs of colorectalcancer patients is used. With respect to each of the NK cells culturedin a CO₂ incubator according to Examples 1, 2 and Comparative Examples1, 2, on Day 6 of culture in a T 25 culture flask, cells are inoculatedinto a 350 mL bag on the basis of the cell number of 1.0×10⁵ to2.0×10⁶/mL and further cultured for 4 days. On Day 10 of culture, thecells are inoculated into a 1 L bag on the basis of the cell number of1.0×10⁵ to 2.0×10⁶/mL and then further cultured for 4 days. Finally, onDay 14 of culture, the cells are inoculated into a 1 L bag on the basisof the cell number of 1.0×10⁵ to 2.0×10⁶/mL and then further culturedfor 3 to 6 days.

The colorectal cancer patients are grouped randomly and marked. Thecontrol group is not injected with NK cells. The NK cell-treated (+IL-2)group is injected three times with 1-3×10⁷ NK cells/per kg of bodyweight and 500 IU/mL of IL-2 at six week intervals intravenously. The NKcell-treated (−IL-2) group is injected six times with 1-3×10⁷ NKcells/per kg of body weight at six week intervals intravenously.

Tumor growth is monitored at 1, 3, 6, 12 months. After 12 months, the NKcell-treated (−IL-2) group exhibits overall decrease in tumor size, andthe NK cell-treated (+IL-2) group exhibits further overall decrease intumor size.

Experimental Example 14. Confirmation of Anticancer Effect of NK Cellsin Alzheimer's Disease Patients

CD56+NK cells are produced according to the method of Examples 1, 2 andComparative Examples 1, 2 for 18 days, except that PBMCs of colorectalcancer patients is used. With respect to each of the NK cells culturedin a CO₂ incubator according to Examples 1, 2 and Comparative Examples1, 2, on Day 6 of culture in a T 25 culture flask, cells are inoculatedinto a 350 mL bag on the basis of the cell number of 1.0×10⁵ to2.0×10⁶/mL and further cultured for 4 days. On Day 10 of culture, thecells are inoculated into a 1 L bag on the basis of the cell number of1.0×10⁵ to 2.0×10⁶/mL and then further cultured for 4 days. Finally, onDay 14 of culture, the cells are inoculated into a 1 L bag on the basisof the cell number of 1.0×10⁵ to 2.0×10⁶/mL and then further culturedfor 3 to 6 days.

Alzheimer's Disease patients are grouped randomly and marked. Thecontrol group is not injected with NK cells. The NK cell-treated groupis injected six times with 1-3×10⁷ NK cells/per kg of body weight and500 IU/mL of IL-2 at weekly intervals intravenously.

Cognitive function of the patient are monitored at 1, 3, 6, 12 months.After 12 months, the NK cell-treated group exhibits improved cognitivefunction.

Experimental Example 15. Confirmation of Anticancer Effect of NK Cellsin Autoimmune Disease Patients

CD56+NK cells are produced according to the method of Examples 1, 2 andComparative Examples 1, 2 for 18 days, except that PBMCs of colorectalcancer patients is used. With respect to each of the NK cells culturedin a CO₂ incubator according to Examples 1, 2 and Comparative Examples1, 2, on Day 6 of culture in a T 25 culture flask, cells are inoculatedinto a 350 mL bag on the basis of the cell number of 1.0×10⁵ to2.0×10⁶/mL and further cultured for 4 days. On Day 10 of culture, thecells are inoculated into a 1 L bag on the basis of the cell number of1.0×10⁵ to 2.0×10⁶/mL and then further cultured for 4 days. Finally, onDay 14 of culture, the cells are inoculated into a 1 L bag on the basisof the cell number of 1.0×10⁵ to 2.0×10⁶/mL and then further culturedfor 3 to 6 days.

Multiple sclerosis patients are grouped randomly and marked. The controlgroup is not injected with NK cells. The NK cell-treated group isinjected six times with 1-3×10 NK cells/per kg of body weight and 500IU/mL of IL-2 at weekly intervals intravenously.

Cognitive function of the patient are monitored at 1, 3, 6, 12 months.After 12 months, the NK cell-treated group exhibits improved cognitivefunction.

Terminology

The foregoing description of the exemplary embodiments has beenpresented only for the purposes of illustration and description and isnot intended to be exhaustive or to limit the invention to the preciseforms disclosed. Many modifications and variations are possible in lightof the above teaching. It is contemplated that various combinations orsub combinations of the specific features and aspects of the embodimentsdisclosed above may be made and still fall within one or more of theinventions. Further, the disclosure herein of any particular feature,aspect, method, property, characteristic, quality, attribute, element,or the like in connection with an embodiment can be used in all otherembodiments set forth herein. Accordingly, it should be understood thatvarious features and aspects of the disclosed embodiments can becombined with or substituted for one another in order to form varyingmodes of the disclosed inventions. Thus, it is intended that the scopeof the present inventions herein disclosed should not be limited by theparticular disclosed embodiments described above. Moreover, while theinvention is susceptible to various modifications, and alternativeforms, specific examples thereof have been shown in the drawings and areherein described in detail. It should be understood, however, that theinvention is not to be limited to the particular forms or methodsdisclosed, but to the contrary, the invention is to cover allmodifications, equivalents, and alternatives falling within the spiritand scope of the various embodiments described and the appended claims.Any methods disclosed herein need not be performed in the order recited.The methods disclosed herein include certain actions taken by apractitioner, however, they can also include any third-party instructionof those actions, either expressly or by implication. The rangesdisclosed herein also encompass any and all overlap, sub-ranges, andcombinations thereof.

The embodiments were chosen and described in order to explain theprinciples of the invention and their practical application so as toactivate others skilled in the art to utilize the invention and variousembodiments and with various modifications as are suited to theparticular use contemplated. Alternative embodiments will becomeapparent to those skilled in the art to which the invention is definedby the appended claims rather than the foregoing description and theexemplary embodiments described therein.

Conditional language, such as “can,” “could,” “might,” or “may,” unlessspecifically stated otherwise, or otherwise understood within thecontext as used, is generally intended to convey that certainembodiments include, while other embodiments do not include, certainfeatures, elements, and/or steps. Thus, such conditional language is notgenerally intended to imply that features, elements, and/or steps are inany way required for one or more embodiments.

The terms “comprising,” “including,” “having,” and the like aresynonymous and are used inclusively, in an open-ended fashion, and donot exclude additional elements, features, acts, operations, and soforth. Also, the term “or” is used in its inclusive sense (and not inits exclusive sense) so that when used, for example, to connect a listof elements, the term “or” means one, some, or all of the elements inthe list.

The ranges disclosed herein also encompass any and all overlap,sub-ranges, and combinations thereof. Language such as “up to,” “atleast,” “greater than,” “less than,” “between,” and the like includesthe number recited.

Numbers preceded by a term such as “approximately”, “about”, and“substantially” as used herein include the recited numbers (e.g., about10%=10%), and also represent an amount close to the stated amount thatstill performs a desired function or achieves a desired result. Forexample, the terms “approximately”, “about”, and “substantially” mayrefer to an amount that is within less than 10% of, within less than 5%of, within less than 1% of, within less than 0.1% of, and within lessthan 0.01% of the stated amount.

The term “generally” as used herein represents a value, amount, orcharacteristic that predominantly includes or tends toward a particularvalue, amount, or characteristic. As an example, in certain embodiments,the term “generally uniform” refers to a value, amount, orcharacteristic that departs from exactly uniform by less than 20%, lessthan 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, andless than 0.01%.

The ranges disclosed herein also encompass any and all overlap,sub-ranges, and combinations thereof. Language such as “up to,” “atleast,” “greater than,” “less than,” “between” and the like includes thenumber recited. Numbers preceded by a term such as “about” or“approximately” include the recited numbers. For example, “about 5.0 cm”includes “5.0 cm.”

Some embodiments have been described in connection with schematicdrawings. However, it should be understood that the schematic drawingsare not drawn to scale. Distances are merely illustrative and do notnecessarily bear an exact relationship to actual dimensions and layoutof the devices illustrated.

For purposes of this disclosure, certain aspects, advantages, and novelfeatures are described herein. It is to be understood that notnecessarily all such advantages may be achieved in accordance with anyparticular embodiment. Thus, for example, those skilled in the art willrecognize that the disclosure may be embodied or carried out in a mannerthat achieves one advantage or a group of advantages as taught hereinwithout necessarily achieving other advantages as may be taught orsuggested herein.

Moreover, while illustrative embodiments have been described herein, thescope of any and all embodiments having equivalent elements,modifications, omissions, combinations (e.g., of aspects across variousembodiments), adaptations and/or alterations as would be appreciated bythose in the art based on the present disclosure. The limitations in theclaims are to be interpreted broadly based on the language employed inthe claims and not limited to the examples described in the presentspecification or during the prosecution of the application, whichexamples are to be construed as non-exclusive. Further, the actions ofthe disclosed processes and methods may be modified in any manner,including by reordering actions and/or inserting additional actionsand/or deleting actions. It is intended, therefore, that thespecification and examples be considered as illustrative only, with atrue scope and spirit being indicated by the claims and their full scopeof equivalents.

1-23. (canceled)
 24. A method of producing natural killer cells,comprising: co-culturing PBMCs with a combination of feeder cells in thepresence of IL-2 and IL-21.
 25. The method of claim 24, furthercomprising isolating at least one of CD56+ cells and/or CD3−/CD56+ cellsand/or CD3− from the PBMCs, which is conducted by using at least one ofCD56 microbeads and CD3 microbeads.
 26. The method of claim 24, whereinthe combination of feeder cells comprises irradiated Jurkat cells andirradiated Epstein-Barr virus transformed lymphocyte continuous line(EBV-LCL) cells.
 27. The method of claim 26, wherein the ratio of theirradiated Jurkat cells and the irradiated EBV-LCL cells is about1:0.1-5 or 0.1-5:1.
 28. The method of claim 26, wherein each of theirradiated Jurkat cells and the irradiated EBV-LCL cells are obtained byirradiation of 50-500 Gy.
 29. The method of claim 26, wherein theco-culturing comprises co-culturing for 1-50 days.
 30. The method ofclaim 26, further comprising: co-culturing the PBMCs cells with acombination of feeder cells, in the presence of IL-2 for a first period;and subsequently co-culturing the PBMCs with the combination of feedercells, in the presence of IL-21 for a second period.
 31. The method ofclaim 26, wherein the IL-21 is added once or more during Day 0-6 of thesecond period.
 32. The method of claim 31, wherein the IL-21 and thecombination of feeder cells are added once or more during Day 0-6 of thesecond period.
 33. The method of claim 30, wherein the IL-21 is addedonce or more during the first six days of every fourteen-day cycleduring the second period.
 34. A composition made by the method of claim24.
 35. A composition for treating cancer in a patient in need thereof,comprising: an effective amount of CD56+ natural killer cells derivedfrom peripheral blood, wherein the effective amount is in a range ofabout 1×10⁶ to 5×10⁸ cells per kg of the patient's body weight, andwherein the CD56+ natural killer cells are at least about 90% pure; IL-2having a concentration of 50-50,000 IU/mL; and a pharmaceuticallyacceptable carrier.
 36. A method of making a therapeutic composition,the method comprising: co-culturing PBMCs with a combination of feedercells in the presence of one or more cytokines to produce an expanded NKcell population; and storing the expanded NK cell population in astorage solution that comprises IL-2, wherein the IL-2 is present in anamount sufficient to inhibit apoptosis of the expanded NK cellpopulation, wherein the expanded NK cell population is stored for atleast 48 hours, and wherein the expanded NK cell population is at leastabout 90% pure.
 37. A method of treating a human subject in need of cellimmunotherapy, comprising: storing at least a therapeutically effectiveamount of an expanded NK cell population in a storage solution thatcomprises IL-2, wherein the IL-2 is present in an amount sufficient toinhibit apoptosis of the expanded NK cell population, wherein theexpanded NK cell population comprises CD56+ cells and/or CD3−/CD56+cells, and wherein the expanded NK cell population is stored for atleast 48 hours; and administering the therapeutically effective amountof the expanded NK cell population to the human subject.
 38. A method oftreating a cancer, comprising: administering a therapeutic compositionto a patient, the therapeutic composition comprising: an effectiveamount of at least one of CD56+ and/or CD3−/CD56+ natural killer cellsderived from peripheral blood mononuclear cells (PBMCs), wherein theeffective amount is in a range of about 1×10⁶ to 5×10⁸ cells per kg ofthe patient's body weight, and wherein the natural killer cells are atleast about 90% pure; IL-2 having a concentration of 50-50,000 IU/mL;and a pharmaceutically acceptable carrier.
 39. A method of treating acancer, comprising: administering a therapeutic composition to a patientwho has cancer, the therapeutic composition comprising having beenprepared by co-culturing PBMCs with a combination of feeder cells in thepresence of IL-2 at a concentration of 50-1000 IU/ml and IL-21 at aconcentration of 10-100 ng/ml.
 40. A method of expanding human naturalkiller cells in culture, comprising: isolating at least one of humanCD56+ cells and/or human CD3−/CD56+ cells from the human PBMCs; andco-culturing the at least one of human CD56+ cells and/or humanCD3−/CD56+ cells with a combination of human feeder cells in thepresence of at least two human cytokines; wherein the combination offeeder cells comprises irradiated Jurkat cells and irradiatedEpstein-Barr virus transformed lymphocyte continuous line (EBV-LCL)cells, wherein the at least two cytokines comprise IL-2 and IL-21. 41.The method of claim 1, IL-2 is present at a concentration of 50-1000IU/ml and IL-21 is present at a concentration of 10-100 ng/ml.